The present invention relates to an anti-heparanase antibody and, more particularly, to a heparanase activity neutralizing monoclonal anti-heparanase antibody.
Heparan sulfate proteoglycans (HSPGs): HSPGs are ubiquitous macromolecules associated with the cell surface and the extracellular matrix (ECM) of a wide range of cells of vertebrate and invertebrate tissues (1-5). The basic HSPG structure consists of a protein core to which several linear heparan sulfate chains are covalently attached. The polysaccharide chains are typically composed of repeating hexuronic and D-glucosamine disaccharide units that are substituted to a varying extent with N- and O-linked sulfate moieties and N-linked acetyl groups (1-5). Studies on the involvement of ECM molecules in cell attachment, growth and differentiation revealed a central role of HSPGs in embryonic morphogenesis, angiogenesis, metastasis, neurite outgrowth and tissue repair (1-5). The heparan sulfate (HS) chains, unique in their ability to bind a multitude of proteins, ensure that a wide variety of effector molecules cling to the cell surface (4-6). HSPGs are also prominent components of blood vessels (3). In large vessels they are concentrated mostly in the intima and inner media, whereas in capillaries they are found mainly in the subendothelial basement membrane where they support proliferating and migrating endothelial cells and stabilize the structure of the capillary wall. The ability of HSPGs to interact with ECM macromolecules such as collagen, laminin and fibronectin, and with different attachment sites on plasma membranes suggests a key role for this proteoglycan in the self-assembly and insolubility of ECM components, as well as in cell adhesion and locomotion. Cleavage of HS may therefore result in disassembly of the subendothelial ECM and hence may play a decisive role in extravasation of normal and malignant blood-borne cells (7-9). HS catabolism is observed in inflammation, wound repair, diabetes, and cancer metastasis, suggesting that enzymes that degrade HS play important roles in pathologic processes.
Involvement of heparanase in tumor cell invasion and metastasis: Circulating tumor cells arrested in the capillary beds of different organs must invade the endothelial cell lining and degrade its underlying basement membrane (BM) in order to escape into the extravascular tissue(s) where they establish metastasis (10). Several cellular enzymes (e.g., collagenase IV, plasminogen activator, cathepsin B, elastase, etc.) are thought to be involved in degradation of the BM (10). Among these enzymes is an endo-xcex2-D-glucuronidase (heparanase) that cleaves HS at specific intrachain sites (7, 9, 11-12). Expression of a HS degrading heparanase was found to correlate with the metastatic potential of mouse lymphoma (11), fibrosarcoma and melanoma (9) cells. The same is true for human breast, bladder and prostate carcinoma cells (U.S. patent application Ser. No. 09/071,739 now U.S. Pat. No. 6,177,545). Moreover, elevated levels of heparanase were detected in sera (9) and urine (U.S. patent application Ser. No. 09/071,739) of metastatic tumor bearing animals and cancer patients and in tumor biopsies (12). Treatment of experimental animals with heparanase inhibitors such as laminarin sulfate, markedly reduced ( greater than 90%) the incidence of lung metastases induced by B16 melanoma, Lewis lung carcinoma and mammary adenocarcinoma cells (8, 9, 13), indicating that inhibition of heparanase activity by neutralizing antibodies, when available, may be applied to inhibit tumor cell invasion and metastasis.
Possible involvement of heparanase in tumor angiogenesis: It was previously demonstrated that heparanase may not only function in cell migration and invasion, but may also elicit an indirect neovascular response (15). These results suggest that the ECM HSPGs provide a natural storage depot for bFGF and possibly other heparin-binding growth promoting factors. Heparanase mediated release of active bFGF from its storage within ECM may therefore provide a novel mechanism for induction of neovascularization in normal and pathological situations (6, 18).
Expression of heparanase by cells of the immune system: Heparanase activity correlates with the ability of activated cells of the immune system to leave the circulation and elicit both inflammatory and autoimmune responses. Interaction of platelets, granulocytes, T and B lymphocytes, macrophages and mast cells with the subendothelial ECM is associated with degradation of heparan sulfate (HS) by heparanase activity (7). The enzyme is released from intracellular compartments (e.g., lysosomes, specific granules, etc.) in response to various activation signals (e.g., thrombin, calcium ionophore, immune complexes, antigens, mitogens, etc.), suggesting its regulated involvement and presence in inflammatory sites and autoimmune lesions. Heparan sulfate degrading enzymes released by platelets and macrophages are likely to be present in atherosclerotic lesions (16). Treatment of experimental animals with heparanase inhibitors markedly reduced the incidence of experimental autoimmune encephalomyelitis (EAE), adjuvant athritis and graft rejection (7, 17) in experimental animals, indicating that the use of neutralizing antibodies to inhibit heparanase activity may inhibit autoimmune and inflammatory diseases (7, 17).
Use of monoclonal antibodies for clinical therapeutics: monoclonal antibodies (Mabs) are beginning to gain a prominent role in the therapeutics arena. Approximately 80 Mabs are in clinical development which represent over 30% of all biological proteins undergoing clinical trials (20, 24). Market entry of new Mab therapies is expected to be dramatically accelerated. Fueling this growth has been the emergence of technologies to create increasingly human-like (humanized) Mabs, ranging from chimerics to fully human. These new Mabs promise to overcome the human antibody to mouse antibody response (25).
Monoclonal antibodies, which can be viewed as nature""s own form of xe2x80x9crational drug designxe2x80x9d, can offer an accelerated drug-discovery approach for appropriate targets. That is because producing high affinity Mabs that specifically block the activity of an antigen target is -usually easier and faster than designing a small molecule with similar activity (23).
Due to their long serum half-life, low toxicity and high specificity, Mabs began to reveal their true therapeutic potential, particularly in oncology, where current therapeutic regimens have toxic side effects that, in many cases, require repetitive dosing in the respective treatment protocols (23).
Until today only two therapeutic Mabs have been approved for sale in the U.S.xe2x80x94the mouse OKT-3 for prevention of organ transplant rejection, and the mouse-human chimeric Fab fragment, for prevention of acute cardiac ischemia following coronary angioplasty (25). Recently, Herceptin, humanized Mab raised against the protooncogene HER-2/neu, has passed Phase III clinical tests in treating breast cancer patients with metastatic disease (23).
In using anti-angiogenesis approach in preventing metastasic disease, Genetech introduced a recombinant humanized Mab to the vascular endothelial growth factor (VEGF). The anti-VEGF rhu Mab was found to be safe and well tolerated in a 25-patient pilot Phase I clinical study (23).
There is thus a widely recognized need for, and it would be highly advantageous to have a heparanase activity anti-heparanase monoclonal antibody.
It is one object of the present invention to provide heparanase activity neutralizing monoclonal anti-heparanase antibody for inhibition of heparanase activity and a method of preparing same.
It is another object of the present invention to provide a pharmaceutical composition including the antibody for treating a condition associated with heparanase activity, thus to use the antibody as a therapeutic agent.
Thus, according to one aspect of the present invention there is provided a monoclonal antibody elicited by a heparanase protein or an immunogenical portion thereof, the monoclonal antibody specifically inhibits heparanase activity.
According to another aspect of the present invention there is provided a pharmaceutical composition comprising as an active ingredient thereof a monoclonal antibody elicited by a heparanase protein or an immunogenical portion thereof, the monoclonal antibody specifically inhibits heparanase activity, and a pharmaceutical acceptable carrier.
According to further features in preferred embodiments of the invention described below, the heparanase protein or the immunogenical portion thereof is recombinant.
According to still further features in the described preferred embodiments the heparanase activity is of recombinant heparanase or natural heparanase.
According to still further features in the described preferred embodiments the monoclonal antibody is humanized.
According to still further features in the described preferred embodiments at least about 90% of the heparanase activity is abolished by the inhibition when about 1 to about 1-40 ratio of heparanase to antibody is realized.
According to still further features in the described preferred embodiments the monoclonal antibody binds to a Cxe2x80x2-terminal portion of heparanase.
According to still another aspect of the present invention there is provided an in vivo or in vitro method of preparing a heparanase activity neutralizing monoclonal anti-heparanase antibody, the method comprising the steps of (a) exposing cells capable of producing antibodies to a heparanase protein or an immonogenical part thereof and thereby generating antibody producing cells; (b) fusing the antibody producing cells with myeloma cells and thereby generating a plurality of hybridoma cells each producing monoclonal antibodies; and (c) screening the plurality of monoclonal antibodies to identify a monoclonal antibody which specifically inhibits heparanase activity.
According to still further features in the described preferred embodiments the method further comprising the step of humanizing the heparanase activity neutralizing monoclonal anti-heparanase antibody.
According to still further features in the described preferred embodiments the immonogenical part of the heparanase protein includes a Cxe2x80x2-terminal portion of the heparanase protein.
According to still another aspect of the present invention there is provided a method of treating a condition associated with heparanase expression comprising the step of administering a heparanase activity neutralizing monoclonal anti-heparanase antibody, the monoclonal anti-heparanase antibody being for neutralizing heparanase catalytic activity.
According to still further features in the described preferred embodiments the condition is associated with altered function of a HSPG associated biological effector molecule.
According to still further features in the described preferred embodiments the HSPG associated biological effector molecule is selected from the group consisting of a growth factor, a chemokine, a cytokine and a degradative enzyme.
According to still further features in the described preferred embodiments the growth factor is selected from the group consisting of HGH, FGF and VEGF.
According to still further features in the described preferred embodiments the chemokine is selected from the group consisting of PF-4, IL-8, MGSA, IP-10, NAP-2, MCP-1, MIP-1xcex1, MIP-1xcex2 and RANTES.
According to still further features in the described preferred embodiments the cytokine is selected from the group consisting of IL-3, TNFxcex1, TNFxcex2, GM-CSF and IFNxcex3.
According to still further features in the described preferred embodiments the degradative enzyme is selected from the group consisting of elastase, lipoprotein lipase and cathepsin G.
According to still further features in the described preferred embodiments the condition is selected from the group consisting of angiogenesis, cell proliferation, tumor cell proliferation, invasion of circulating tumor cells, metastases, inflammatory disorders and autoimmune conditions.
The present invention successfully addresses the shortcomings of the presently known configurations by providing a heparanase activity neutralizing monoclonal anti-heparanase antibody, method for its preparation, identification and characterization, pharmaceutical composition including same and the use of same for treating various medical conditions.